This invention relates to immunosuppression and more particularly provides monoclonal antibodies and other binding molecules against the CD25 antigen.
In organ transplant surgery, particularly kidney, liver, heart, lung and bone marrow transplant surgery, it is necessary to suppress the immune system of the graft recipient to minimise the likelihood of graft rejection after surgery. Various immunosuppressive drugs have been proposed for this purpose but their use has to be carefully controlled since, in addition to undesirable side-effects arising from the use of certain immunosuppressive agents, there is also the difficulty that the immunosuppressive action makes the graft recipient particularly susceptible to infection by bacteria and viruses that would be controlled by a normal immune system. Immunosuppressive agents that have been used successfully in clinical practice include steroids, azathioprine and cyclosporin A. It is necessary in clinical practice to attempt to balance the degree of immunosuppression necessary to prevent or treat graft rejection episodes with the retention of a certain amount of the recipient""s immune system to combat other infectious agents and, at the same time, to keep any possible undesirable side-effects under control.
In addition to the use of immunosuppressive drugs, attention has also focused upon the use of certain monoclonal antibodies (MAbs) to suppress immune reactions, in particular, attention has been paid to monoclonal antibodies that recognise various surface antigens of T-cells. Here too, problems have been encountered in clinical practice, namely that prior art antibodies were either too powerful or not sufficiently effective, and sometimes caused severe side effects such as high fever.
These MAb""s are generally designated by a CD (Cluster Determination) number assigned by successive Leucocyte Typing Workshops. Although a term such as CD3 is now frequently applied to the cell surface antigen, and a MAb to this antigen is often described as xe2x80x9canti-CD3xe2x80x9d, in the following description terms such as CD3, CD25 etc. will be applied to MAb""s and the corresponding cell surface antigens will be described as xe2x80x9cCD3 antigenxe2x80x9d etc.
In particular, monoclonal antibodies to membrane antigens present on all T-cells (also called pan T-cell antigens) such as the CD3 antigen are very potent antibodies in that they have an overall suppressive activity on the immune system. Therefore, the human body may be deprived of the immediate immune response usually mediated by the memory T-cells once an infection occurs. This is certainly not desirable when attempting to prevent rather than to cure graft rejection episodes. A treatment suitable for use in prophylaxis should be essentially selective, i.e. the pool of memory T-cells should be kept intact while the category of T-cells (activated T-cells) which could be directly involved in a rejection event should be inactivated.
This desirable goal may be achieved using antibodies to activated T-cells. These T-cells are characterised by the presence of the high affinity IL-2 receptor on their membrane surface. The high affinity IL-2 receptor is composed of at least two different polypeptide chains, an xcex1-chain also known as the CD25 antigen, and a xcex2-chain. Resting T-cells do not express this high affinity receptor but low and intermediate affinity receptors which consist of xcex1- or xcex2-chain homodimers. A CD25 antibody which interferes with the binding of IL-2 to its high affinity receptor and therefore selectively suppresses the immune response, is an antibody of choice for the prophylaxis of graft rejection episodes.
Natural immunoglobulins or antibodies comprise a generally Y-shaped multimeric molecule having an antigen-binding site at the end of each upper arm. The remainder of the structure, in particular the stem of the Y mediates effector functions associated with the immunoglobulins. The general structure of an antibody of the IgG class is shown schematically in FIG. 1A. Both heavy and light chains comprise a variable domain and a constant part. An antigen binding site consists of the variable domain of a heavy chain associated with the variable domain of a light chain. The variable domains of the heavy and light chains have the same general structure which is illustrated in FIG. 1B.
More particularly, the antigen binding characteristics of an antibody are essentially determined by 3 specific regions in the variable domain of the heavy and light chains which are called hypervariable regions or complementarity determining regions (CDRs). As shown in FIG. 1B, these 3 hypervariable regions alternate with 4 framework regions, (FRs) whose sequences are relatively conserved and which are not directly involved in binding. The CDRs form loops and are held in close proximity by the framework regions which largely adopt a xcex2-sheet conformation. The CDRs of a heavy chain together with the CDRs of the associated light chain essentially constitute the antigen binding site of the antibody molecule.
The determination as to what constitutes an FR or a CDR region is usually made by comparing the amino acid sequence of a number of antibodies raised in the same species. The general rules for identifying the CDR and FR regions are given in Table C.
Furthermore, it has been recently found that the contribution made by a light chain variable domain to the energetics of binding is small compared to that made by the associated heavy chain variable domain and that isolated heavy chain variable domains have an antigen binding activity on their own. Such molecules are now commonly referred to as single domain antibodies.
Several murine CD25 MAbs already exist and include 33B3-1 (Immunotech-Merieux), BDxcex1IL-2R (Becton-Dickinson), 2C8 (Amersham), Campath 6 (MRC, Cambridge) and ATH207 (free University, Berlin). However, it has now been found that a novel mouse CD25 antibody of the IgG2a isotype, hereinafter called RFT5-IgG2a, has better properties than the CD25 antibodies of the prior art especially with regard to binding affinity, and that it is possible to construct other CD25 binding molecules having the same hypervariable regions as RFT5-IgG2a.
Accordingly, the invention provides a CD25 binding molecule which comprises at least one antigen binding site comprising at least one domain which comprises in sequence, the hypervariable regions CDR1, CDR2 and CDR3; said CDR1 having the amino acid sequence Arg-Tyr-Trp-Met-His (SEQ. ID. NO: 7), said CDR2 having the amino acid sequence Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly (SEQ. ID. NO: 8), and said CDR3 having the amino acid sequence Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe (SEQ. ID. NO: 9); and direct equivalents thereof.
In a first aspect of the invention, the CD25 binding molecule comprises a single antigen binding site comprising a single domain.
In a second aspect of the invention, the CD25 binding molecule comprises at least one antigen binding site comprising:
a) a first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3; said CDR1 having the amino acid sequence Arg-Tyr-Trp-Met-His (SEQ. ID. NO: 7), said CDR2 having the amino acid sequence Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly (SEQ. ID. NO: 8), and said CDR3 having the amino acid sequence Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe (SEQ. ID. NO: 9) and,
b) a second domain comprising in sequence the hypervariable regions CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2, said CDR1xe2x80x2 having the amino acid sequence Ser-Ala-Ser-Ser-Ser-Ile-Ser-Tyr-Met-Gln (SEQ. ID. NO: 10), said CDR2xe2x80x2 having the amino acid sequence Asp-Thr-Ser-Lys-Leu-Ala-Ser (SEQ. ID. NO: 11), and said CDR3xe2x80x2 having the amino acid sequence His-Gln-Arg-Ser-Ser-Tyr-Thr (SEQ. ID. NO: 12);
and direct equivalents thereof.
Unless otherwise indicated, any polypeptide chain is hereinafter described as having an amino acid sequence starting at the N-terminal extremity and ending at the C-terminal extremity.
When the antigen binding site comprises both the first and second domains, these may be located on the same polypeptide molecule or, preferably, each domain may be on a different chain, the first domain being part of an immunoglobulin heavy chain or fragment thereof and the second domain being part of an immunoglobulin light chain or fragment thereof.
By xe2x80x9cCD25 binding moleculexe2x80x9d is meant any molecule capable of binding to the CD25 antigen either alone or associated with other molecules to form high affinity IL-2 receptors. The binding reaction may be shown by standard methods (qualitative assays) including, for example, a bioassay for determining the inhibition of IL-2 binding to its receptor or any kind of binding assays, with reference to a negative control test in which an antibody of unrelated specificity, e.g. an anti-lysosyme antibody, is used. Advantageously, the binding of the molecule of the invention to the CD25 antigen may be shown in a competitive binding assay using the AHT207, BDxcex1IL-2-R or 33B3-1 antibody as competitors. Preferably, the AHT207 or BDxcex1IL-2-R antibody will be chosen as competitors. A particular example of a binding assay is given below.
Human peripheral blood mononuclear cells (HPBM) are grown in culture medium RPMI 1640 supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 xcexcg/ml streptomycin, 25 mM sodium bicarbonate and 10% fetal calf serum (FCS). 1 xcexcg/ml phytohemagglutinin (PHA) is used to stimulate HPBM. After 3 days, the blasts are resuspended at a concentration of 3.106/ml in phosphate buffered saline supplemented with 2% bovine serum albumin (BSA) and 2% azide. 50 xcexcl samples of this suspension are incubated for 10 mn, at 20xc2x0 C., under non-capping conditions, with graded concentrations of a blocking antibody (competitor) from 1 to 100 xcexcg/ml. Then 1 xcexcg/ml of biotinylated antibody of the invention is added to the cells and the incubation is continued for 10 min. Cells are washed and further incubated for 10 min with fluorescein-labelled streptavidin. Cells are again washed, fixed with formalin and analysed with a fluoro-cytometer which detects the binding of the biotinylated antibody. In parallel, an experiment is carried out with a biotinylated antibody of an unrelated specificity, as a negative control.
Examples of antigen binding molecules include antibodies as produced by B-cells or hybridomas and chimeric or humanized antibodies or any fragment thereof, e.g. F(abxe2x80x2)2 and Fab fragments, as well as single chain or single domain antibodies.
A single chain antibody consists of the variable domains of an antibody heavy and light chains covalently bound by a peptide linker usually consisting of from 10 to 30 amino acids, preferably from 15 to 25 amino acids. Therefore, such a structure does not include the constant part of the heavy and light chains and it is believed that the small peptide spacer should be less antigenic than a whole constant part. By xe2x80x9cchimeric antibodyxe2x80x9d is meant an antibody in which the constant regions of heavy or light chains or both are of human origin while the variable domains of both heavy and light chains are of non-human (e.g. murine) origin. By xe2x80x9chumanized antibodyxe2x80x9d is meant an antibody in which the hypervariable regions (CDRs) are of non-human (e.g. murine) origin, while all or substantially all the other parts of the immunoglobulin e.g. the constant regions and the highly conserved parts of the variable domains, i.e. the framework regions, are of human origin. A humanized antibody may however retain a few amino acids of the murine sequence in the parts of the framework regions adjacent to the hypervariable regions.
Hypervariable regions may be associated with any kind of framework regions, preferably of murine or human origin. Suitable framework regions are described in xe2x80x9cSequences of proteins of immunological interestxe2x80x9d, Kabat E. A. et al, US department of health and human services, Public health service, National Institute of Health. However, the preferred heavy chain framework is that of RFT5-IgG2a, which is shown in Table A and Seq. Id. Nos: 1 to 3. It consists in sequence of FR1, FR2, FR3 and FR4 regions. In a similar manner, Table B and Seq. Id. Nos. 4 to 6 shows the preferred RFT5-IgG2a light chain framework which consists, in sequence, of FR1xe2x80x2, FR2xe2x80x2, FR3xe2x80x2 and FR4xe2x80x2 regions.
Accordingly, the invention also provides a CD25 binding molecule which comprises at least one antigen binding site comprising either a first domain having an amino acid sequence substantially identical to that shown in Seq. Id. No. 1 starting with the amino acid at position 1 and ending with the amino acid at position 117 or a first domain as described above and a second domain having an amino acid sequence substantially identical to that shown in Seq. Id. No. 4, starting with amino acid at position 1 and ending with the amino acid at position 104.
Monoclonal antibodies raised against a protein naturally found in all humans must necessarily be developed in a non-human system e.g. in mice. As a direct consequence of this, a xenogenic antibody as produced by a hybridoma, when administered to humans, elicits an undesirable immune response which is predominantly mediated by the constant part of the xenogenic immunoglobulin. This clearly limits the use of such antibodies as they cannot be administered over a prolonged period of time. Therefore it is particularly preferred to use single chain, single domain, chimeric or humanized antibodies which are not likely to elicit a substantial allogenic response when administered to humans.
In view of the foregoing, a more preferred CD25 binding molecule of the invention is selected from a chimeric anti-CD25 antibody which comprises at least
a) one immunoglobulin heavy chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3 and (ii) the constant part or fragment thereof of a human heavy chain; said CDR1 having the amino acid sequence Arg-Tyr-Trp-Met-His (SEQ. ID. NO: 7), said CDR2 having the amino acid sequence Ala-Ile-Tyr-Pro-Gly-Asn-Ser-Asp-Thr-Ser-Tyr-Asn-Gln-Lys-Phe-Glu-Gly (SEQ. ID. NO: 8), and said CDR3 having the amino acid sequence Asp-Tyr-Gly-Tyr-Tyr-Phe-Asp-Phe (SEQ. ID. NO: 9) and
b) one immunoglobulin light chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2 and (ii) the constant part or fragment thereof of a human light chain; said CDR1xe2x80x2 having the amino acid sequence Ser-Ala-Ser-Ser-Ser-Ile-Ser-Tyr-Met-Gln (SEQ. ID. NO: 10), said CDR2xe2x80x2 having the amino acid sequence Asp-Thr-Ser-Lys-Leu-Ala-Ser (SEQ. ID. NO: 11), and said CDR3xe2x80x2 having the amino acid sequence His-Gln-Arg-Ser-Ser-Tyr-Thr (SEQ. ID. NO: 12);
and direct equivalents thereof.
Alternatively, a CD25 binding molecule of the invention may be selected from a single chain binding molecule which comprises an antigen binding site comprising
a) a first domain comprising in sequence the hypervariable regions CDR1, CDR2 and CDR3, said hypervariable regions having the amino acid sequences as shown in Table A and Seq. Id. Nos. 1 to 3,
b) A second domain comprising in sequence the hypervariable regions CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2, said hypervariable regions having the amino acid sequences as shown in Table B and Seq. Id. Nos. 4 to 6 and
c) a peptide linker which is bound either to the N-terminal extremity of the first domain and to the C-terminal extremity of the second domain or to the C-terminal extremity of the first domain and to the N-terminal extremity of second domain;
and direct equivalents thereof.
As it is well known, minor changes in an amino acid sequence such as deletion, addition or substitution of one or several amino acids may lead to an allelic form of the original protein which has substantially identical properties. Thus, by the term xe2x80x9cdirect equivalents thereofxe2x80x9d is meant either any single domain CD25 binding molecule (molecule X)
(i) in which the hypervariable regions CDR1, CDR2 and CDR3 taken as a whole are at least 80% homologous, preferably at least 90% homologous, more preferably at least 95% homologous to the hypervariable regions as shown in Seq. Id. No. 3 and,
(ii) which is capable of inhibiting the binding of IL-2 to its receptor substantially to the same extent as a reference molecule having framework regions identical to those of molecule X but having hypervariable regions CDR1, CDR2 and CDR3 identical to those shown in Seq. Id. No. 3;
or any CD25 binding molecule having at least two domains per binding site (molecule Xxe2x80x2)
(i) in which the hypervariable regions CDR1, CDR2, CDR3, CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2 taken as a whole are at least 80% homologous, preferably at least 90% homologous, more preferably at least 95% homologous to the hypervariable regions as shown in Seq. Id. Nos. 3 and 6 and
(ii) which is capable of inhibiting the binding of IL-2 to its receptor substantially to the same extent as a reference molecule having framework regions and constant parts identical to molecule Xxe2x80x2 but having hypervariable regions CDR1, CDR2, CDR3, CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2 identical to those shown in Seq. Id. Nos. 3 and 6.
This last criterion may be conveniently tested in various assays including a Mixed Lymphocyte Reaction (MLR) bioassay, an antigen specific HPBM response bioassay and an IL-2 dependent T lymphoblast proliferation bioassay. Such assays are described hereinafter in the text. By the term xe2x80x9cto the same extentxe2x80x9d is meant that the reference and the equivalent molecules exhibit, on a statistical basis, essentially identical IL-2 binding inhibition curves in one of the bioassays referred to above.
Most preferably, the chimeric CD25 antibody comprises at least
a) one heavy chain which comprises a variable domain having an amino acid sequence substantially identical to that shown in Seq. Id. No. 1 starting with glutamic acid at position 1 and ending with serine at position 117 and the constant part of a human heavy chain; and
b) one light chain which comprises a variable domain having an amino acid sequence substantially identical to that shown in Seq. Id. No. 4 starting with glutamine acid at position 1 and ending with lysine acid at position 104 and the constant part of a human light chain.
The constant part of a human heavy chain may be of the xcex31, xcex32, xcex33, xcex34, xcexc, xcex11, xcex12, xcex4 or xcex5 type, preferably of the xcex3 type, more preferably of the xcex31 type, whereas the constant part of a human light chain may be of the xcexa or xcex type (which includes the xcex1, xcex2 and xcex3 subtypes) but is preferably of the xcexa type. The amino acid sequence of all these constant parts are given in Kabat et al (Supra).
Conjugates of the CD25 binding molecules of the invention, e.g. enzyme or toxin or radioisotope conjugates, are also included within the scope of the invention.
A CD25 binding molecule of the invention may be produced by recombinant DNA techniques. In view of this, one or more DNA molecules encoding the binding molecule must be constructed, placed under appropriate control sequences and transferred into a suitable host organism for expression.
In a very general manner, there are accordingly provided
(i) DNA molecules encoding a single domain CD25 binding molecule, of the invention, a single chain CD25 binding molecule of the invention, a heavy or light chain or fragments thereof of a CD25 binding molecule of the invention and
(ii) the use of the DNA molecules of the invention for the production of a CD25 binding molecule of the invention by recombinant means.
The present state of the art is such that the skilled man will be able to synthetize the DNA molecules of the invention given the information provided herein i.e. the amino acid sequences of the hypervariable regions and the DNA sequences coding for them. A method for constructing a variable domain gene is for example described in EPA 239 400 and may be briefly summarized as follows: A gene encoding a variable domain of a MAb of whatever specificity is cloned. The DNA segments encoding the framework and hypervariable regions are determined and the DNA segments encoding the hypervariable regions are removed so that the DNA segments encoding the framework regions are fused together with suitable restriction sites at the junctions. The restriction sites may be generated at the appropriate positions by mutagenesis of the DNA molecule by standard procedures. Double stranded synthetic CDR cassettes are prepared by DNA synthesis according to the sequences given in Seq. Id. No. 1 or 4. These cassettes are provided with sticky ends so that they can be ligated at the junctions of the framework. A protocol for achieving a DNA molecule encoding an immunoglobulin variable domain is shown in FIG. 5.
Furthermore, it is not necessary to have access to the mRNA from a producing hybridoma cell line in order to obtain a DNA construct coding for the MAbs of the invention. Thus PCT application WO 90/07861 gives full instructions for the production of a MAb by recombinant DNA techniques given only written information as to the nucleotide sequence of the gene. The method comprises the synthesis of a number of oligonucleotides, their amplification by the PCR method, and their splicing to give the desired DNA sequence.
Expression vectors comprising a suitable promoter or genes encoding heavy and light chain constant parts are publicly available. Thus, once a DNA molecule of the invention is prepared it may be conveniently transferred in an appropriate expression vector. DNA molecules encoding single chain antibodies may also be prepared by standard methods, for example, as described in WO 88/1649.
In view of the foregoing, and since the mouse MAb as naturally secreted by the hybridoma is not the preferred type of MAb, it is believed that no hybridoma deposit is necessary to comply with the criteria of sufficiency of description.
In a particular embodiment of the invention, the recombinant means for the production of a CD25 binding molecule includes first and second DNA constructs as described below:
The first DNA construct encodes a heavy chain or fragment thereof and comprises
a) a first part which encodes a variable domain comprising alternatively framework and hypervariable regions, said hypervariable regions being in sequence CDR1, CDR2 and CDR3 the amino acid sequences of which are shown in Seq. Id. No. 1; this first part starting with a codon encoding the first amino acid of the variable domain and ending with a codon encoding the last amino acid of the variable domain, and
b) a second part encoding a heavy chain constant part or fragment thereof which starts with a codon encoding the first amino acid of the constant part of the heavy chain and ends with a codon encoding the last amino acid of the constant part or fragment thereof, followed by a non-sense codon.
Preferably, this first part encodes a variable domain having an amino acid sequence substantially identical to the amino acid sequence as shown in Seq. Id. No. 1 starting with the amino acid at position 1 and ending with the amino acid at position 117. More preferably the first part has the nucleotide sequence as shown in Seq. Id. No. 1 starting with the nucleotide at position 142 and ending with the nucleotide at position 492. Also preferably, the second part encodes the constant part of a human heavy chain, more preferably the constant part of the human xcex31 chain. This second part may be a DNA fragment of genomic origin (comprising introns) or a cDNA fragment (without introns).
The second DNA construct encodes a light chain or fragment thereof and comprises
a) a first part which encodes a variable domain comprising alternatively framework and hypervariable regions; said hypervariable regions being in sequence CDR1xe2x80x2, CDR2xe2x80x2 and CDR3xe2x80x2, the amino acid sequences of which are shown in Seq. Id. No. 4; this first part starting with a codon encoding the first amino acid of the variable domain and ending with a codon encoding the last amino acid of the variable domain, and
b) a second part encoding a light chain constant part or fragment thereof which starts with a codon encoding the first amino acid of the constant part of the light chain and ends with a codon encoding the last amino acid of the constant part or fragment thereof followed by a non-sense codon.
Preferably, this first part encodes a variable domain having an amino acid sequence substantially identical to the amino acid sequence as shown in Seq. Id. No. 4 starting with the amino acid at position 1 and ending with the amino acid at position 104. More preferably, the first part has the nucleotide sequence as shown in Seq. Id. No. 4 starting with the nucleotide at position 244 and ending with the nucleotide at position 555 Also preferably the second part encodes the constant part of a human light chain, more preferably the constant part of the human xcexa chain.
In the first and second DNA constructs, the first and second parts are preferably separated by an intron. In the intron located between the first and second part, an enhancer is preferably inserted. The presence of this genetic element which is transcribed but not translated, may be required for an efficient transcription of the second part. More preferably the first and second DNA constructs comprise the enhancer of a heavy chain gene advantageously of human origin.
The first or second DNA construct advantageously comprises a third part which is located upstream of the first part and which encodes part of a leader peptide; this third part starting with the codon encoding the first amino acid and ending with the last amino acid of the leader peptide. This peptide is required for secretion of the chains by the host organism in which they are expressed and is subsequently removed by the host organism. Preferably, the third part of the first DNA construct encodes a leader peptide having an amino acid sequence substantially identical to the amino acid sequence as shown in Table A, starting with the amino acid at position xe2x88x9219 and ending with the amino acid at position xe2x88x921. Also preferably, the third part of the second DNA construct encodes a leader peptide having an amino acid sequence as shown in Table B, starting with the amino acid at position xe2x88x9222 and ending with the amino acid at position xe2x88x921.
Each of the DNA constructs are placed under the control of suitable control sequences, in particular under the control of a suitable promoter. Any kind of promoter may be used, provided that it is adapted to the host organism in which the DNA constructs will be transferred for expression. However, if expression is to take place in a mammalian cell, it is particularly preferred to use the promoter of an immunoglobulin gene.
The desired antibody may be produced in a cell culture or in a transgenic animal. A suitable transgenic animal may be obtained according to standard methods which include micro injecting into eggs the first and second DNA constructs placed under suitable control sequences, transferring the so prepared eggs into appropriate pseudo-pregnant females and selecting a descendant expressing the desired antibody.
When the antibody chains have to be produced in a cell culture, the DNA constructs must first be inserted into either a single expression vector or into two separate but compatible expression vectors, the latter possibility being preferred.
Accordingly, the invention also provides an expression vector able to replicate in a prokaryotic or eukaryotic cell line which comprises at least one of the DNA constructs above described.
Each expression vector containing a DNA construct is then transferred into a suitable host organism. When the DNA constructs are separately inserted on two expression vectors, they may be transferred separately, i.e. one type of vector per cell, or co-transferred, this latter possibility being preferred. A suitable host organism may be a bacterium, a yeast or a mammalian cell line, this latter being preferred. More preferably, the mammalian cell line is of lymphoid origin e.g. a myeloma, hybridoma or a normal immortalized B-cell, but does not express any endogeneous antibody heavy or light chain.
It is also preferred that the host organism contains a large number of copies of the vectors per cell. If the host organism is a mammalian cell line, this desirable goal may be reached by amplifying the number of copies according to standard methods. Amplification methods usually consist of selecting for increased resistance to a drug, said resistance being encoded by the expression vector.
In another aspect of the invention, there is provided a process for producing a multi-chain CD25 binding molecule which comprises (i) culturing an organism which is transformed with first and second DNA constructs of the invention and (ii) recovering an active CD25 binding molecule from the culture.
Alternatively, the heavy and light chains may be separately recovered and reconstituted into an active binding molecule after in vitro refolding. Reconstitution methods are well-known in the art; Examples of methods are in particular provided in EPA 120 674 or in EPA 125 023.
Therefore a process may also comprise
(i) culturing a first organism which is transformed with a first DNA construct of the invention and recovering said heavy chain or fragment thereof from the culture and
(ii) culturing a second organism which is transformed with a second DNA construct of the invention and recovering said light chain or fragment thereof from the culture and
(iii) reconstituting in vitro an active CD25 binding molecule from the heavy chain or fragment thereof obtained in (i) and the light chain or fragment thereof obtained in (ii).
In a similar manner, there is also provided a process for producing a single chain or single domain CD25 binding molecule which comprises (i) culturing an organism which is transformed with a DNA construct respectively encoding a single chain or single domain CD25 binding molecule of the invention and (ii) recovering said molecule from the culture.
CD25 binding molecules of the invention exhibit very good immunomodulatory activity as shown, for example, in a mixed lymphocyte reaction (MLR) bioassay (Akbar et al, J. Immunol. 140, 2171-8). The MLR is generally considered to be the in vitro equivalent of the allogeneic transplant response which leads to rejection in vivo.
1. Inhibition of the MLR
From a HPBM preparation of a first donor are aliquoted 100 xcexcl samples containing 105 HPBM to which are added various concentrations of a molecule of the invention ranging from 0 to 300 ng/ml (including these limiting values). Then each sample is mixed with a 100 xcexcl aliquot containing 105 HLA-incompatible X-irradiated HPBM of a second donor, or T-cell depleted HPBM. The mixture is incubated for 6 days at 37xc2x0 C., and 1 xcexcCi of methyl 3H-thymidine (3H-Tdr) in 10 xcexcl volume is then added. After 6 hours, the cell proliferation is measured by radioactivity incorporation.
In this particular assay, the molecules of the invention show an in vitro immunomodulatory activity at concentrations of from 0.3 ng/ml as shown in FIG. 6. 50% of the cellular growth is inhibited at about 3 ng/ml.
The immunomodulatory activity of the molecules of the invention may also be estimated by measuring the inhibition of antigen-specific HPBL response or the inhibition of IL-2 dependent T-lymphoblast proliferation as follows:
2. Inhibition of Antigen-specific HPBM Response
The molecules of the invention inhibit efficiently the generation of a PPD (tuberculin) specific, HLA class II restricted T-cell response, indicating their ability to inhibit the binding of endogenously produced IL-2 to its receptor. In vivo these antigen specific responses are expected to play a crucial role in the initiation of autoimmunity and transplantation rejection.
From a preparation of HPBM are aliquoted 100 xcexcl samples containing 105 HPBM to which are added various concentrations of a molecule of the invention ranging from 0 to 300 ng/ml (including these limiting values) and tuberculin (PPD) at a final concentration of 30 xcexcg/ml. The samples are incubated for 6 days at 37xc2x0 C., and 1 xcexcCi of methyl 3H-thymidine is then added in a 10 xcexcl volume. After 6 hours of incubation, cell proliferation is measured by radioactivity incorporation.
In this particular assay, the molecules of the invention show an immunomodulatory activity of from about 10 ng/ml as shown in FIG. 7. 50% of the cellular growth is inhibited at about 50 ng/ml.
3. Inhibition of IL-2 Dependent T-lymphoblast Proliferation
The molecules of the invention inhibit efficiently the IL-2 dependent growth of human T cell blasts induced by MLR or PPD stimulation. These cells are expected to play a major role in the chronicity of autoimmunity and rejection episodes.
Triplicate cultures containing 20xc3x97103 5 day old PPD or MLR stimulated HPBM in a final volume of 200 xcexcl are incubated at 37xc2x0 C. for 48 hours in the presence of 5, 10 or 20 ng/ml recombinant IL-2 and a molecule of the invention at various concentrations ranging from 0 to 10 xcexcg/ml (including these limiting values). Then 3H-Tdr is added. After 6 hours, cell proliferation is measured by radioactivity incorporation. In this particular assay, the molecules of the invention show an immunomodulatory activity at concentrations of from 0.1 xcexcg/ml as shown in FIGS. 8A and 8B, 8C and 8D.
Therefore the invention also provides
(i) the use of a CD25 binding molecule of the invention in immunosuppression of a human immune system
(ii) a method of immunosuppressing the human immune system which comprises administering an immunosuppressive effective amount of a CD25 binding molecule of the invention to a patient in need of such treatment.
(iii) a pharmaceutical composition for immunosuppressing the human immune system which comprises a CD25 binding molecule of the invention and a pharmaceutically acceptable carrier or diluent.
In particular, a CD25 binding molecule of the invention is useful for preventing or treating graft rejection episodes.
For these indications, the appropriate dosage will, of course, vary depending upon, for example, the particular molecule of the invention to be employed, the host, the mode of administration and the nature and severity of the condition being treated. However, in prophylactic use, satisfactory results are generally indicated to be obtained at daily dosages from about 0.1 mg to about 1 mg per kilogram body weight. These dosages should be increased by up to a factor of 4 for treating a rejection event when it actually occurs. A molecule of the invention is conveniently administered parenterally, normally intravenously, for example, into the antecubital or other peripheral vein. A prophylactic treatment typically comprises administering the molecule of the invention once daily to once weekly for 2 to 4 weeks, starting on the day of transplantation, preferably some hours before transplantation.
The molecules of the invention may also be useful in the treatment of malignancies of cells expressing the CD25 antigen, for example in the treatment of T-cell leukemia and certain other leukemias and lymphomas. For this purpose, the CD25 binding molecule may be used in the form of a radioconjugate in which the molecule is coupled to an alpha-emitting radionuclide.
The molecules of the invention may also be useful in the treatment or prophylaxis of HIV infection. It appears that the HIV virus requires proliferating T cells in order to multiply, and thus inhibition of T cell proliferation by blocking the CD25 antigen should also inhibit the multiplication of the virus.
Pharmaceutical compositions of the invention may be manufactured in conventional manner. A composition according to the invention is preferably provided in lyophilized form. For immediate administration it is dissolved in a suitable aqueous carrier, for example sterile water for injection or sterile buffered physiological saline. If it is considered desirable to make up a solution of larger volume for administration by infusion rather as a bolus injection, it is advantageous to incorporate human serum albumin or the patient""s own heparinised blood into the saline at the time of formulation. The presence of an excess of such physiologically inert protein prevents loss of monoclonal antibody by adsorption onto the walls of the container and tubing used with the infusion solution. If albumin is used, a suitable concentration is from 0.5 to 4.5% by weight of the saline solution.
According to a further aspect of the invention, it has been found that particularly beneficial results can be achieved by the use, in combination, of at least two antigen binding molecules to activated T-cells, said binding molecules recognizing at least two different antigens characteristic of activated T-cells.
Preferably a combination of two different antigen binding molecules is used, each recognizing a different antigen. Thus although both antigen binding molecules recognize activated T-cell surface antigens, they are not competing with each other for the same binding site on activated T-cells.
Preferably one of the antigen binding molecules is a CD25 binding molecule.
Accordingly the present invention also provides an immuno suppressive composition comprising a mixture of at least one CD25 binding molecule and at least one antigen binding molecule to at least one antigen other than CD25 which is characteristic of activated T-cells.
The invention further provides at least two antigen binding molecules to activated T-cells in association with one another for use in immunosuppression of the mammalian system, said antigen binding molecules recognizing at least two different antigens characteristic of activated T-cells, one of which is the CD25 antigen.
By xe2x80x9cantigen binding molecule to activated T-cellsxe2x80x9d is meant a binding molecule which strongly reacts with activated T-cells while it reacts weakly or not at all with resting T-cells. Preferably the antigen binding molecules are complete immunoglobulin molecules, more preferably murine, chimeric, or humanized monoclonal antibodies, particularly chimeric monoclonal antibodies. The preferred CD25 monoclonal antibodies are those having CDR""s with the amino acid sequences described above.
Advantageously, the composition of the invention may also include or may be used in combination with an immunosuppressive drug such as cyclosporin A.
The preferred monoclonal antibodies to activated T-cell antigens other than CD25 are typically those classified in the CD7 cluster as established by the Boston Workshop and reported in xe2x80x9cLeucocyte Typing II, Vol. I human T lymphocytesxe2x80x9d by Reinherz, Haynes, Nadler and Berstein, Springer Verlag, 1985. The CD7 antigen is heterogeneously expressed on about 80% of resting T-cells. However, the expression strongly increases upon activation (a 2-3 fold rise in intensity).
A preferred combination of antibodies is therefore a combination of a CD7 with a CD25 antibody. Accordingly, the composition of the invention preferably comprises a mixture of at least one CD25 antibody together with at least one CD7 antibody, more preferably of one CD25 antibody together with one CD7 antibody. Also preferably, both antibodies are of the IgG isotype.
The two antibodies, optionally together with an immunosuppressive drug, can be used in clinical practice in various ways. Preferably they are mixed together and the physical mixture is administered to the patient. An alternative procedure is the administration of the antibodies and optionally the immunosuppressive drug to the recipient from separate reservoirs in any order but at the same time. The composition may be prepared and administered parenterally as described above for the single CD25 antibody. Alternatively, the immunosuppressive drug is administered orally and the monoclonal antibodies are administered parenterally, separately or as a mixture.
To aid in making up suitable compositions, the monoclonal antibodies and optionally an immunosuppressive drug, may be packaged separately within the same container, with instructions for mixing or concomitant administration. Examples of kits include for example a multi-barrelled syringe or a twin pack containing separate unit dose forms of at least two antibodies to activated T-cells, said anti-bodies recognizing at least two different antigens characteristic of activated T-cells, one of which is the CD25 antigen.
Investigations so far indicate that the administration of the antibodies in combination with one another and optionally with an immunosuppressive drug is free from unacceptable side-effects at the dosage levels employed and that there is no potentiation of the side-effects observed with the individual antibodies. For use in prophylaxis, a suitable dosage will normally call for the administration of the order of 0.05-0.5 milligram of a first antibody (such as the CD25 antibody) per kilogram body weight of the patient and 0.05-0.5 milligram of a second antibody (such as a CD7 antibody) per kilogram body weight. When the immunosuppressive drug is cyclosporin, the recommended amount of the immunosuppressive drug which can be optionally used is 2 to 5 milligram per kilogram body weight when administered parenterally and 10-15 mg/kilogram body weight when administered orally. The composition of the invention may be administered on a daily or weekly basis, preferably on a weekly basis.
Although the composition of the invention is particularly designed for use in prophylaxis of graft rejection episodes, its use can be conveniently extended to the treatment of rejection events when they actually occur. In this case, the dosages should be increased by up to a factor of 4.
Murine monoclonal antibodies suitable for use in the present invention are known per se. Many monoclonal antibodies against activated T-cell surface antigens are available from Culture Collections in various countries of the world and specifically, the American Type Culture Collection of Rockville, Md., USA can provide suitable monoclonal antibodies or hybridomas secreting such antibodies. An example of hybridoma secreting CD7 monoclonal antibodies that can be used in the present invention and that is available from the ATCC is T3-3A1. Other CD7 antibodies are RFT-2 and CHH 380 (a chimeric antibody). CD25 antibodies include, besides the preferred RFT-5 and its chimeric derivative as described above; M7/2 (Gaulton et al, Clin. Immunol. and Immunopath. (1985) 36: 18); the anti-tac antibody (Uchiyama et al, J. Immunol. (1981) 126 (4): 1393); and the Campath 6 monoclonal antibody.
The synergistic effect of a combination of CD25 and CD7 monoclonal antibodies is demonstrated in vitro by the MLR bioassay described above, and also in vivo in clinical tests on human patients.
In the MLR bioassay, inhibition of the 3H-TdR uptake is observed in cultures to which a CD7 (RFT2) or a CD25 (RFT5) monoclonal antibody are added singly, and there is a substantially greater degree of inhibition when both of these antibodies are used together at the same total concentration. The MLR is the in vitro equivalent of the allogeneic transplant response which leads to rejection in vivo while the inhibition described above is equivalent to immunosuppression in vivo.
In MLRs to which cyclosporin is added over a dose range from 10 nanograms/ml to 100 xcexcg/ml, in the presence of CD7 or CD25 monoclonal antibodies there is an increased inhibition of 3H-TdR compared to cyclosporin alone over the whole dose range. The combination of CD7, CD25 and cyclosporin shows a greater inhibitory effect than any other combination.
In clinical tests, patients about to undergo kidney, liver or heart transplantation are selected for prophylactic therapy. On the day of transplantation, 2 hours before surgery, a first intravenous infusion of the chimeric CD25 antibody of Example 5 together with chimeric CD7 antibody (CHH 380) is administered at a dose of 0.2 mg of each antibody per kg of body weight. Two days after surgery an identical infusion of the two antibodies at 0.4 mg/kg of body weight is administered and then repeated at weekly intervals for one month.
The intravenous infusions are prepared as follows: the lyophylized antibodies are mixed together and dispersed into 100 ml sterile buffered saline containing 4.5% wt. of human albumin. This saline dispersion is administered to the patients over a 30 minute period. The patients also receive standard cyclosporin therapy. No patients undergo a rejection episode during the one month therapy period.